Immunoglobulins (Igs), or antibodies, as the main effectors of humoral immunity and endogenous modulators of the immune response, have proven to be valuable molecules in clinical applications such as diagnostic, therapeutic and prophylactic purposes, as well as in research contexts. An immunoglobulin molecule is composed of four protein chains; two heavy and two light chains which must be produced, associate and correctly fold to be effectively exported from the cell. The heavy and light chains are held together covalently through disulfide bonds. The heavy chains are also covalently linked via disulfide bonds in a base portion often referred to as the constant region. This constant region is also responsible for a given immunoglobulin molecule being mutually recognizable with certain sequences found at the surface of particular cells or factors. There are five known major classes of constant regions (IgG, IgM, IgA, IgD and IgE) which determine the class, or effector function, of the immunoglobulin molecule.
The cDNA of the heavy chain is composed of a leader sequence (a signal sequence) (approximately 57 bp/19 aa) which is removed upon maturation of the protein, a variable region, VH (approximately 350 bp/115 aa), and the constant region, CH (approximately 990 bp/330 aa). The cDNA of the light chain is composed of a leader sequence (approximately 66 bp/22 aa) which is removed upon maturation of the protein; a variable region, Vκ or Vλ (approximately 350 bp/115 aa); and a constant region, Cκ or Cλ (approximately 321 bp/107 aa). The light chains covalently bind to the N-terminal Y branches of the two heavy chains. The variable regions of the heavy and light chains, approximately 110–125 amino acids in length, confer specificity of particular antigenic epitopes for a particular immunoglobulin molecule.
Although the antibody genes can be engineered to be expressed in bacteria and plants such as corn, effective expression of antibodies should be carried out using mammalian systems in order to obtain efficient mammalian post-translational modifications and folding processes required for effective assembly and secretion of functional antibody molecules. Presently used methods for production of antibodies include in vivo immunization followed by collection and processing in order to obtain purified antibody. However, such methods are limited in that they may require large amounts of antigen to produce response, and they are time-consuming processes. Furthermore, antigen may not generate effective response in the animal, and generation of therapeutically useful antibodies is questionable. Therefore the time and effort expended to produce antibodies using such present methods may not culminate in sufficient antibody production.
Additional methods for production of antibodies involve creation of expression systems for use in mammalian cells lines, such as CHO or murine myeloma cell lines. See, McCafferty, J., et al. Eds. Antibody Engineering, A Practical Approach. IRL Press, (1997). Present methods for development of antibody expression systems involve creation of a vector composed of a light chain flanked by its promoter and a poly-adenylation (polyA) region in a plasmid. Also, a heavy chain vector composed of a heavy chain construct flanked by a promoter and a polyA region is created. The promoter/insert/polyA region of the heavy chain may then alternatively be combined into the light chain vector in order to create a single vector containing both heavy and light chain molecules. In these methods, genomic DNA or mRNA of a heavy or light chain is cloned into an expression vector for use in mammalian cell lines or transgenic animals. Another method of construction includes vectors which are created wherein a framework of the immunoglobulin chain (e.g., heavy chain sequence) is inserted prior to addition of particular variable sequences as desired, such as has been disclosed and described in U.S. Pat. No. 5,780,225.
Currently utilized methods of production of immunoglobulin expression systems, however, require multiple cloning steps resulting in production of only a single desired molecule. If alterations in specificity (governed by the variable region) or effector function (governed by the constant region) of the antibody are desired, re-assembly of a new light and/or heavy chain vector, followed by re-assembly of a final combined vector is required.
It thus would be desirable to provide new immunoglobulin molecule constructs useful as effectors of humoral immunity and modulators of immune responses, as well as methods for effective production of such molecules. It would be particularly desirable to provide such constructs and methods that would facilitate production of antibodies having desired specificity in comparison to methods of the prior art. It also would be desirable to provide such constructs and methods that allow for facilitated production of immunoglobulins having desired effector functions as compared to those of the prior art. Such molecules and methods preferably would be simple in construction and require straightforward assembly steps in order to obtain the desired immunoglobulins, thereby resulting in rapid generation of desired antibody molecules with comparatively little effort. Furthermore, development of a system resulting in large scale production in comparison to present methods will be beneficial for development and production of protein therapeutics.